Response of gene expression in zebrafish exposed to pharmaceutical mixtures: Implications for environmental risk
Graphical abstract
Introduction
Pharmaceutical substances are constantly released to the aquatic environment principally through municipal effluents and wastewater treatment plants leading to chronic exposure in aquatic organisms (Brain et al., 2004, Halling-Sørensen et al., 1998, Jones et al., 2005, Nakada et al., 2006). Discharges of these substances are likely to increase in the future because of increases in their production, human population growth, and demographic shifts towards higher proportions of older people that use greater amounts of pharmaceuticals (Daughton and Ternes, 1999). Although present at low concentrations at the ng L−1 to µg L−1 range (Gómez et al., 2007, Gros et al., 2007, Ternes, 1998, Thomas and Foster, 2004), pharmaceutical substances are found simultaneously as complex mixtures that have unknown and difficult to evaluate effects on aquatic biota (Blasco and DelValls, 2008, Gagné et al., 2006, Lara-Martín et al., 2014, Yoon et al., 2010).
It is possible that individual substances can act in a synergistic or additive manner, which suggests management for environmental protection should take into consideration mixtures of substances rather than models based on individual effects. Pharmaceuticals are an example of substances for which additive effects on toxicity in aquatic organisms have been observed (Cleuvers et al., 2003; Christensen et al., 2007; Henry and Black, 2007). Pharmaceuticals may induce cytochrome P450 enzyme activity (CYP1A) by binding to the aryl hydrocarbon receptor (AhR). The induction of CYP1A1 gene transcription by the aryl hydrocarbon begins by their binding and activating the AhR, a cytosolic protein that, on ligand binding, translocates to the nucleus and with its partner, the aryl hydrocarbon nuclear translocator, interacts with the promoter of the CYP1A1 gene (Rowlands and Gustafsson, 1997). This results in an up-regulation of transcription and a subsequent increase in CYP1A1 mRNA and enzyme levels. Consequently, this mechanism is reflected in the rated of deethylation of the substrate 7-ethoxyresorufin by cytochrome P450 (CYP) to give the product resorufin. The 7-ethoxyresorufin O-deethylase (EROD) activity is a biomarker used to determine AhR agonist exposure to certain polyhalogenated aromatic hydrocarbons (PHAHs) and polycyclic aromatic hydrocarbons (PAHs) (Bucheli and Fent, 1995, Gokøyr and Förlin, 1992) Responses of the cytochrome P450 system are used as biomarkers of oxidative stress in fish (Bucheli et al., 1995). The Induction of CYP1A by pharmaceuticals may generate reactive oxygen species (ROS) (Van der Berg et al., 1998). When a substrate is metabolized by cytochrome P450 consumes one molecule of molecular oxygen leading to an oxidized substrate plus a molecule of water as a by-product. However, for most CYPs, depending on the nature of the substrate, the reaction is "uncoupled", consuming more O2 than the metabolized substrate and producing activated oxygen or O2- (Gonzalez and Tukey, 2006).
It has been demonstrated that mixtures of substances that induce cyp1a a gene that encodes for CYP1A, the induction is consistent with the addition of the individual induction activities of each substance (Hook et al., 2008, Filby et al., 2007). Induction of cyp1a and the activity of CYP1A are important because increased generation of ROS can lead to oxidative stress and damage to biomolecules, or abnormally enhance the CYP1A metabolism of endogenous substrates (Rifkind, 2006). It is also considered of greatest concern for risk assessment pharmaceuticals that bind the estrogen and induce estrogenic effects in fish such as oral contraceptives, hormone replacement therapies, motor deficits associated with menopause, hypoestrogenism, and the management of some pre- and postmenopausal symptoms (Laurenson et al., 2014). In particular, mixtures of estrogenic substances can cause effects [e.g., induction of vitellogenin genes (vtg) or vitellogenin lipoprotein (Vg) production] in fish that are reported to be equivalent to the addition of their individual activities (Filby et al., 2007, Thorpe et al., 2006, Thorpe et al., 2001). In addition, even when concentrations of individual substances in a mixture are below levels that cause estrogenic effects as single substances, their combined effect within a mixture can be sufficient to induce estrogenicity in fish (Brian et al., 2005, Ketan and Collins, 2007). Consequently, analysis of the potential for substances to influence fish vitellogenin (vtg or VG) is frequently used as a biomarker of estrogenic activity (Filby et al., 2007). Although numerous studies have examined toxicological impacts of pharmaceuticals, the biological activity of these substances in non-target organisms remain uncertain, and interactions among pharmaceuticals when present in mixtures can be considerably more complicated than that of additive toxicity based on a single mechanism of action. The toxicity of pharmaceutical mixtures in fish has been investigated at various levels of biological organization and results have not yet provided a clear direction on how management of this environmental issue should be approached. At issue is whether management of complex mixtures can be addressed by understanding effects of individual substances and predicting fish responses to complex mixtures, or whether each possible mixture combination leads to a unique organism response that must be assessed independently.
The objective of the present research is to investigate whether effects of individual substances on expression of target genes (vtg and cyp1a) in zebrafish larvae can be used to predict the change in expression when fish are exposed to substance mixtures. To support observations on changes in cyp1a expression, evaluation of CYP1A enzymatic activity was assessed in parallel by evaluation of ethoxyresorufin O-deethylase (EROD) activity. Pharmaceuticals from different therapeutic groups (Table 1) were selected for this study based on their frequency of use, presence as mixtures in surface waters, and concentrations in municipal effluents (Table 2). Caffeine (Caf) is a potent stimulant of the central nervous system (Nikolau et al., 2007) and has been used as a marker for residual wastewater in the environment due to its persistance. The anti-inflammatory non-prescription drug ibuprofen (Ibu) is used as an analgesic and antipyretic and, in addition to naproxen, is one of the most abundant anti-inflammatory drugs found in municipal effluents (Miège et al., 2009; Stuer-Lauridsen et al., 2000). Carbamazepine (Cbz) is a psychiatric drug prescribed as an anticonvulsant and mood-stabilizer applied in the treatment of epilepsy, bipolar disorder, and trigeminal neuralgia (García-Morales et al., 2007). Tamoxifen (Tmx) is one of the most commonly used chemotherapeutic agents, has anti-estrogenic activity (Bergh, 2003, Osborne, 1998, Powles et al., 1994), and has been detected in many waste water treatment plant effluents (Table 2) (Lara-Martín et al., 2014, Roberts and Thomas, 2006).
Section snippets
Experimental fish
Zebrafish embryos were obtained from the Zebrafish Research Facility (ZRF) housed at Plymouth University, Plymouth, UK. All experiments were conducted in this facility with appropriate approval from the UK Home Office. In all experiments, zebrafish larvae were treated humanely and with regard for alleviation of suffering. Larvae were routinely bred from bulk spawning of stock fish. Water quality in the ZRF was measured and water used in these experiments (fish water) had the following
Fish mortality
No mortality was observed in larvae exposed to control or to the solvent control (0.001% EtOH). Mortality increased with increasing phenanthrene concentration, the LC50 was 0.57 mg L−1, and all fish died at a concentration of 1 mg L−1 (Fig. 1a). This experiment was used to identify sub-lethal phenanthrene concentrations for assessment of cyp1a expression (phenanthrene was used as positive control), and lethality results were consistent with previous reports of acute toxicity of phenanthrene [LC50
Conclusion
Complex mixtures of pharmaceutical substances can have unpredictable effects on toxicity endpoints than those observed by the individual substances. In this study additive toxicity was not supported, and results highlight the need to evaluate mixtures of pharmaceuticals when performing an environmental risk assessment of drugs, rather than studies based on individual effects, since in the environment pharmaceutical active compounds are not found in isolation and the effects of their mixtures is
Acknowledgements
Authors would like to thank Stan McMahon for fish care and maintenance in Zebrafish Research Facility at Plymouth University. This work was conducted under the framework of the project P09-RNM-5136 from Andalusian Government (Spain). G.A.M. would like to thank the financial support from Consejería de Economía, Innovación y Ciencia from the Regional Government of Andalusia (Spain), Fondos FEDER, Becas Chile (Chilean Government), and to Campus de Excelencia Internacional del Mar (CEIMAR).
References (94)
- et al.
Induction of hepatic CYP1A2 by the oral administration of caffeine to rats: lack of association with the Ah locus
Biochim. Biophys. Acta
(1995) - et al.
CYP1A inhibition in fish gill filaments: a novel assay applied on pharmaceuticals and other chemicals
Aquat. Toxicol.
(2010) - et al.
Effluent from drug manufacturing affects cytochrome P450 1 regulation and function in fish
Chemosphere
(2013) Breast-cancer prevention: is the risk-benefit ratio in favour of tamoxifen?
Lancet
(2003)A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein dye binding
Anal. Biochem.
(1976)- et al.
Microcosm evaluation of the effects of an eight pharmaceutical mixture to the aquatic macrophytes Lemna gibba and Myriophyllum sibiricum
Aquat. Toxicol.
(2004) - et al.
Novel passive dosing system for determining the toxicity of phenanthrene to early life stages of zebrafish
Sci. Total Environ.
(2013) Aquatic ecotoxicity of pharmaceuticals including the assessment of combination effects
Toxicol. Lett.
(2003)- et al.
Seasonal variations in the occurrence and fate of basic and neutral pharmaceuticals in a Swedish river – lake system
Chemosphere
(2010) - et al.
Ecotoxicology of human pharmaceuticals
Aquat. Toxicol.
(2006)
Hepatic metallotionein level and mixed function oxidase activity in fingerling rainbow trout (Oncorhynshus mykiss) after acute exposure to pulp and paper milleffluents
Water Res.
Occurrence of pharmaceuticals products in a municipal effluent and toxicity to rainbow trout (Oncorhynchus mykiss) hepatocytes
Ecotox. Environ. Safe.
The cytochrome P450 1A1 response in fish: application of immunodetection in environmental monitoring and toxicological testing
Mar. Environ. Res.
Pilot survey monitoring pharmaceuticals and related compounds in a sewage treatment plant located on the Mediterranean Coast
Chemosphere
Concentration–response concept in ecotoxicoproteomics: effects of different of different phenanthrene concentrations to the zebrafish (Danio rerio) embryo proteome
Ecotoxicol. Environ. Saf.
Ocurrence, fate and effects of pharmaceuticals substances in the environment - A review
Chemosphere
Changes in the relative expression pattern of multiple vitellogenin genes in adult male and larval zebrafish exposed to exogenous estrogens
Comp. Biochem. Phys. A.
Characterization of genes transcriptionally upregulated in the liver of sand goby (Pomatoschistus minutus) by 17a-ethinyloestradiol: Identification of distinct vitellogenin and zona radiata protein transcripts
Chemosphere
Developmental toxicity of 4-ring polycyclic aromatic hydrocarbons in zebrafish is differentially dependent on AH receptor isoforms and hepatic cytochrome P4501A metabolism
Toxicol. Appl. Pharm.
Effects of short-term exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin on microRNA expression in zebrafish embryos
Toxicol. Appl. Pharm.
The zebrafish gill model: induction of CYP1A, EROD and PAH adduct formation
Aquat. Toxicol.
Acute toxicity of pharmaceutical and personal care products on freshwater crustacean (Thamnocephalus platyurus) and fish (Oryzias latipes)
J. Toxicol. Sci.
Occurrence, distribution and partitioning of nonionic surfactants and pharmaceuticals in the urbanized Long Island Sound Estuary (NY)
Mar. Pollut. Bull.
Effects of human pharmaceuticals on cytotoxicity, EROD activity and ROS production in fish hepatocytes
Toxicology
Simá-Alvarez, R., Gold-Bouchot, G. o, p’-DDT induction of vitellogenesis and its inhibition by tamoxifen in Nile tilapia (Oreochromis niloticus)
Mar. Environ. Res.
CYP1A expression in liver and gill of rainbow trout following waterborne exposure: implications for biomarker determination
Aquat. Toxicol.
Analysis of relative gene expression data using real-time quantitative PCR and the 2(T) (−Delta Delta C) method
Methods
Fate of pharmaceuticals and personal care products in wastewater treatment plants - Conception of a database and first results
Environ. Pollut.
Expression of two vitellogenin genes (vg1 and vg3) in fathead minnow (Pimephales promelas) liver in response to exposure to steroidal estrogens and androgens
Ecotox. Environ. Safe.
Pharmaceutical chemicals and endocrine disrupters in municipal wastewater in Tokyo and their removal during activated sludge treatment
Water Res.
Estrogen-mediated suppression of cytochrome P4501A (CYP1A) expression in rainbow trout hepatocytes: role of estrogen receptor
Chem. Biol. Interact.
Cytochrome P4501A, genotoxic and stress responses in golden grey mullet (Liza aurata) following short-term exposure to phenanthrene
Chemosphere
Fish bioaccumulation and biomarkers in environmental risk assessment: a review
Environ. Toxicol. Phar.
Biomonitoring of aquatic pollution with feral eel (Anguilla anguilla) II. biomarkers: pollution-induced biochemical responses
Aquat. Toxicol.
Multistressor interactions in the zebrafish (Danio rerio): concurrent phenanthrene exposure and Mycobacterium marinum infection
Aquat. Toxicol.
Cobalt-induced genotoxicity in male zebrafish (Danio rerio), with implications for reproduction and expression of DNA repair genes
Aquat. Toxicol.
The occurrence of selected pharmaceuticals in wastewaters effluent and surface waters of lower Tyne catchment
Sci. Total Environ.
Feasibility study of the zebrafish assay as an alternative method to screen for developmental toxicity and embryotoxicity using a training set of 27 compounds
Reprod. Toxicol.
Nitrite-induced enhancement of toxicity of phenanthrene in fish and its implications for coastal waters
Estuar. Coast. Shelf S.
In vitro inhibition of cytochrome P450-mediated reactions by gemfibrozil, erythromycin, ciprofloxacin and fluoxetine in fish liver microsomes
Aquat. Toxicol.
Environmental risk assessment of human pharmaceuticals in Denmark after normal therapeutic use
Chemosphere
Occurrence of drugs in German sewage treatment plants and rivers
Water Res.
Effects of fibrates, anti-inflammatory drugs antidepressants in the fish hepatoma cell line PHLC-1: cytotoxicity and interactions with cytochrome P450 1A
Toxicol. Vitr.
Differential gene expression as a toxicant-sensitive endpoint in zebrafish embryos and larvae
Aquat. Toxicol.
Determination of selected pharmaceuticals and caffeine in sewage and seawater from Tromsø/Norway with emphasis on ibuprofen and its metabolites
Chemosphere
Effects of phenanthrene on hepatic enzymatic activities in tilapia (Oreochromis niloticus ♀ × O. aureus ♂)
J. Environ. Sci.
Occurrence of endocrine disrupting compounds, pharmaceuticals, and personal care products in the Han River (Seoul, South Korea)
Sci. Total Environ.
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